The field of the invention concerns protected amino acid derivatives for solid phase peptide synthesis, namely, N.sub..alpha. -2-(4-nitorphenylsulfonyl)ethoxycarbonyl-amino acids having the general formula I: ##STR2## wherein R.sub.1 represents hydrogen atom, and R.sub.2 may represent hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methlypropyl, tert-butoxymethyl, 1-tert-butoxyethyl, 2-methylthioethyl, benzyl, carboxamidomethyl, 2-carboxamidoethyl, tert-butoxycarbonylmethyl, 2-(tert-butoxycarbonyl)ethyl, 4-(tert-butoxycarbamido)butyl, 4-tert-butoxybenzyl, indolyl-3-methyl, S-(triphenylmethyl)thiomethyl, 1-(triphenylmethyl)imidazolyl-4methyl, 3-(N.sup.G -mesitylenesulfonyl)guanidinopropyl, N-xanthylcarboxamidomethyl, 2-(N-xanthylcarboxamido)ethyl or S-(acetamidomethyl)thiomethyl.
or R.sub.1 and R.sub.2 together represent propylene radical, employed as N.sub..alpha. -protected amino acid derivatives for solid phase peptide synthesis.
Solid phase peptide synthesis is widely employed for the preparation of biologically active peptides which are used in medical and biological research and also as active substances in pharmacy, veterinary and diagnostics.
The essence of solid phase peptide synthesis can be outlined as a stepwise elongation of a peptide chain by means of repeated cycles of chemical reactions, beginning from the first C-terminal amino acid attached to an unsoluble carrier. During the course of the synthesis target products of all reactions remain bound to the carrier, whereas excessive reactants and side-products are removed by filtration and washing of the carrier.
In order to perform the solid phase synthesis of a peptide, the first amino acid (C-terminal of the target amino acid squence) with the protected .alpha.-amino group is linked covalently to an unsoluble polymeric carrier through the free .alpha.-carboxyl group by ester or amide bond formation. Then N.sub..alpha. -protective group is selectively cleaved from thus obtained N.sub..alpha. -protected aminoacyl-polymer, and the aminoacyl-polymer with the free .alpha.-amino group is formed. This polymer is further acylated with the next N.sub..alpha. -protected amino acid, thus giving N.sub..alpha. -protected dipeptidyl-polymer. Such synthetic cycles, which consist of N.sub..alpha. -protection cleavage and of subsequent acylation of free amino group with following N.sub..alpha. -protected amino acid, are repeated until the assembly of target amino acid sequence is completed.
In practical solid phase synthesis large molar excesses(2 to 10-fold) of acylating reagents are usually employed to assure complete conversion, therefore, all reactive groups in side chains of the amino acids, such as amino, carboxyl, hydroxyl, thiol, guanidino groups, should be blocked with appropriate protective groups. The protective groups for this purpose must be selected carefully to provide reliable and permanent protection of the side chains under conditions of peptidyl-polymer acylations and during the cleavage of temporary N.sub..alpha. -protection. On the other hand, these side-chain protective groups must provide the opportunity to deprotect the synthesized peptide in one or two stages quantitatively and without damage of its structure. In most cases the peptidyl-polymer linkage also should be cleaved simultaneously. It is evident that the structure and the chemical properties of permanent protective groups for side-chains of amino acids are determined not only by the nature of reactive function to be protected but in a great extent by the structure and the chemical properties of the employed temporary N.sub..alpha. -protective group. Therefore, temporary N.sub..alpha. -protection is the key element of the whole strategy of solid phase peptide synthesis.
Well known and widely used in solid phase peptide synthesis are N.sub..alpha. -tert-butoxycarbonyl amino acids(Boc-amino acids) described for this purpose by R. B. Merrifield in Biochemistry, 1964, V. 3, p. 1385. tert-Butoxycarbonyl(Boc) group can be cleaved by the action of acidic reagents of medium strength, such as, for example, trifluoroacetic acid and its solutions in chlorinated hydrocarbons, solutions of hydrogen chloride in organic solvents, boron trifluoride/diethyl ether complex and some other acids, with the formation of isobutylene and carbon dioxide.
Together with temporary N.sub..alpha. -Boc-protection for the permanent blocking of side chains protective groups are employed, which are stable during N.sub..alpha. -Boc cleavage but can be cleaved by more strong acidic reagents with the simultaneous fission of peptidyl-polymer bond. Known reagents used for this purpose are liquid hydrogen fluoride, trifuoromethanesulfonic acid and their mixtures with anisole, thioanisole, dimethylsulfide. The main drawback of the synthetic strategy with the use of temporary N.sub..alpha. -Boc-protection is the application of acidolysis for the cleavage of both temporay and permanent protective groups, that cannot provide complete stability of the permanent protection. As the length of synthesized peptide grows, permanent protective groups undergo cumulative action of acidic reagents during Boc cleavage steps, that can result in partial loss of these groups and accumulation of side-products. Apart of this, the final treatment of assemblied peptidyl-polymer with superacidic reagents can cause partial destruction of the target peptide. It also should be mentioned that extremely hazardous propeties of superacids require special equipment and appropriate safety measures during handing.
To aviod the use of superacidic reagents for the final peptide deprotection, several highly acid-sensitive groups were proposed more recently as a temporary N.sub..alpha. -protection, which are considered to be compatible to permanent side-chain protection of so-called tert-butyl type cleavable by acidic reagents of medium strength. An example of such N.sub..alpha. -protective group is 1-(3,5-di-tert-butylphenyl)-1-methyl-ethoxycarbonyl(t-Bumeoc)group, which is described in Collect. Czech. Chem. Commun., 1992, V. 57, p. 1707. N.sub..alpha. -t-Bumeoc-group is cleaved by 1% trifluoroacetic acid in dichloromethane and can be used together with permanent protective groups of t-butyl type cleavable by neat trifluoroacetic acid or its concentrated solutions. In this case the employment of superacids is excluded but general principle of differential acidolysis still remains unchanged.
A different approach to the strategy of solid phase peptide synthesis is outlined by R. B. Merrifield in Science, 1986, V. 232, p. 341. This approach, called "orthogonality principle", is based on the assumption that temporary and permanent protective groups should be removable by totally distinct reagents according to totally distinct chemical mechanisms, so that temporary N.sub..alpha. -protection could be cleaved with absolute selectivity providing full preservation of permanent protection, and vice versa. At present time the "orthogonality principle" is commonly accepted as a guideline for the development of efficient strategies of solid phase peptide synthesis.
As an example of implementation of the "orthogonality principle" the employment of N.sub..alpha. -dithiasuccinylamino acids(Dts-amino acids) in solid phase synthesis is described in Int. J. Peptide and Protein Res., 1987, V. 30. p. 740. N.sub..alpha. -Dithiasuccinyl(Dts) protective group is quite resistant to acidic reagents of medium strength and is cleaved smoothly by thiol reagents in neutral media with the liberation of amino group and formation of carbon thiooxide. Application of Dts-amino acids in practical synthesis is still limited due to the lack of effective methods for their preparation.
The most known and widely employed strategy of solid phase synthesis, which corresponds to the "orthogonality principle", is based on the use of N.sub..alpha. -9-fluorenylmethoxycarbonylamino acids(Fmoc-amino acids), as described by C. D. Chang and J. Meienhofer in Int. J. Peptide and Protein Res., 1975, V. 11, p. 246. N.sub..alpha. -9-Fluorenylmethoxycarbonyl(Fmoc) group is resistant to to acidic reagents and is cleaved according to the .beta.-elimination mechanism by organic bases in aprotic solvents, for example, by morpholine diethylamine, piperazine, or piperidine in dimethylformamide(DMF) or dichloromethane, amino group being liberated and dibenzofulvene together with CO.sub.2 being formed. In solid phase synthesis the cleavage of Fmoc group is preferably performed by the treatment of N.sub..alpha. -protected peptidyl-polymer with 20 to 50% piperidine in DMF during 10 to 30 min. Said conditions allow to use permanent acid sensitive protection of t-butyl type together with temporary N.sub..alpha. -Fmoc-protection, thus providing the "othogonality" of the synthetic strategy.
N.sub..alpha. -Fmoc-amino acids are widely used in manual solid phase peptide syntyesis, as well as in automatic and semi-automatic synthesizer of all types. However, it should be noted that extreme base sensitivity of N.sub..alpha. -Fmoc-protection and some its unstability in neutral aprotic solvents require to control carefully acylation conditions and also the purity of empolyed solvents. Special care sholud be taken when N.sub..alpha. -Fmoc-amino acids are used for the synthesis of peptides exceeding 30 residues in length. Besides, relatively high cost of production prevents the use of Fmoc-derivatives in large scale peptide preparations.